Histones H1 and H4 of surface-spread meiotic chromosomes

The chromatin conformation of somatic and meiotic chromosomes is, at least in part, a function of electrostatic nucleosome interactions that are mediated by transient acetylation of the histone H4 N-terminal domain and phosphorylation of histone H1. The distribution of those histones in the chromatin of meiotic chromosomes is reported here. Antibodies to testis-specific histone 1, H1t, detect H1t in the chromatin of mouse meiotic prophase chromosomes only after synapsis and synaptonemal complex (SC) assembly is completed and before core separation is initiated. The H1t protein is evenly distributed over euchromatin, heterochromatin and the SC. Antibodies to acetylated lysine residues 5, 12 or 16 of histone H4, indicate that the euchromatin is more acetylated than the centromeric heterochromatin. The pattern is most pronounced for acetylated residue 5 and least for 16. Antibodies to phosphorylated H1 epitopes do not react with chromatin but, instead, recognize the chromosome cores and SCs. Possibly these are not phosphorylated histone H1 epitopes, but SC proteins with similar potentially phosphorylatable sequences such as KTPTK of the synaptic protein Syn1.     

黄胫小车蝗卵子发生及卵母细胞凋亡的显微观察

对黄胫小车蝗(Oedaleus infernalis)卵子发生过程和卵母细胞凋亡进行显微观察。结果表明,黄胫小车蝗卵子发生可明显分为3个时期10个阶段,即卵黄发生前期、卵黄发生期和卵壳形成期。第1阶段,卵母细胞位于卵原区,经历减数第一次分裂;第2阶段,卵母细胞核内染色体解体成网状,滤泡细胞稀疏地排列在卵母细胞周围;第3阶段,滤泡细胞扁平状,在卵母细胞周围排成一层;第4阶段,滤泡细胞呈立方形排在卵母细胞周围;第5阶段,滤泡细胞呈长柱形排在卵母细胞周围,滤泡细胞之间、滤泡细胞与卵母细胞之间出现空隙;第6阶段,卵母细胞边缘开始出现卵黄颗粒;第7阶段,卵母细胞中沉积大量卵黄,胚泡破裂;第8阶段,滤泡细胞分泌卵黄膜包围卵黄物质;第9阶段,滤泡细胞分泌卵壳;第10阶段,卵壳分泌结束,卵子发育成熟。卵母细胞发育过程中的凋亡发生在卵黄发生前期,主要表现为滤泡细胞向卵母细胞内折叠,胞质呈团块状等特征。     

Changes in global histone acetylation pattern in somatic cell nuclei after their transfer into oocytes at different stages of maturation

In our study, we have examined the pattern of global histone modification changes in somatic cell nuclei after their transfer into mouse oocytes at different stages of maturation or after their parthenogenetic activation. While germinal vesicle (GV) staged immature oocytes are strongly labeled with anti-acetylated histone H3 and H4 antibodies, the signal is absent in both metaphase I and metaphase II oocytes (MI, MII). In contrast, the oocytes of all maturation stages show a presence of trimethylated H3/K4 in their chromatin. When somatic cells were fused to intact or enucleated GV oocytes, both the GV and the somatic cell nucleus showed a very strong signal for all the antibodies used. On the other hand, when somatic cells nuclei that are AcH3 and AcH4 positive before fusion are introduced into either intact or enucleated MI or MII oocytes, their acetylation signal decreased rapidly and was totally absent after a prolonged culture. This was not the case when anti-trimethyl H3/K4 antibody was used. The somatic cell chromatin showed only a slight decrease in the intensity of labeling after its transfer into MI or MII oocytes. This decrease was, however, evident only after a prolonged culture. These results suggest not only a relatively higher stability of the methylation modification but also some difference between the oocyte and somatic chromatin. The ability to deacetylate the chromatin of transferred somatic nuclei disappears rapidly after the oocyte activation. Our results indicate that at least some reprogramming activity appears in the oocyte cytoplasm almost immediately after GV breakdown (GVBD), and that this activity rapidly disappears after the oocyte activation.     

Distinct acetylation of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> histone H4 during cell cycle,parasite differentiation,and after DNA damage

Histones of trypanosomes are quite divergent when compared to histones of most eukaryotes. Nevertheless, the histone H4 of Trypanosoma cruzi, the protozoan that causes Chagas’ disease, is acetylated in the N terminus at lysines 4, 10, and 14. Here, we investigated the cellular distribution of histone H4 containing each one of these posttranslational modifications by using specific antibodies. Histone H4 acetylated at lysine 4 (H4-K4ac) is found in the entire nuclear space preferentially at dense chromatin regions, excluding the nucleolus of replicating epimastigote forms of the parasite. In contrast, histone H4 acetylated either at K10 or K14 is found at dispersed foci all over the nuclei and at the interface between dense and nondense chromatin areas as observed by ultrastructural immunocytochemistry. The level of acetylation at K4 decreases in nonreplicating forms of the parasites when compared to K10 and K14 acetylations. Antibodies recognizing the K14 acetylation strongly labeled cells at G2 and M stages of the cell cycle. Besides that, hydroxyurea synchronized parasites show an increased acetylation at K4, K10, and K14 after S phase. Moreover, we do not observed specific colocalization of K4 modifications with the major sites of RNA polymerase II. Upon γ-irradiation that stops parasite replication until the DNA is repaired, dense chromatin disappears and K4 acetylation decreases, while K10 and K14 acetylation increase. These results indicate that each lysine acetylation has a different role in T. cruzi. While K4 acetylation occurs preferentially in proliferating situations and accumulates in packed chromatin, K10 and K14 acetylations have a particular distribution probably at the boundaries between packed and unpacked chromatin. Sheila Cristina Nardelli and Julia Pinheiro Chagas da Cunha contributed equally to this work.     

Transition of basic protein during spermatogenesis of <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck, 1765)

According to the ultrastructural characteristic observation of the developing male germ cells, spermatogenesis of the crustacean shrimp, Fenneropenaeus chinensis, is classified into spermatogonia, primary spermatocytes, secondary spermatocyte, four stages of spermatids, and mature sperm. The basic protein transition during its spermatogenesis is studied by transmission electron microscopy of ammoniacal silver reaction and immunoelectron microscopical distribution of acetylated histone H4. The results show that basic protein synthesized in cytoplasm of spermatogonia is transferred into the nucleus with deposition on new duplicated DNA. In the spermatocyte stage, some nuclear basic protein combined with RNP is transferred into the cytoplasm and is involved in forming the cytoplasmic vesicle clumps. In the early spermatid, most of the basic protein synthesized in the new spermatid cytoplasm is transferred into the nucleus, and the chromatin condensed gradually, and the rest is shifted into the pre-acrosomal vacuole. In the middle spermatid, the nuclear basic protein linked with DNA is acetylated and transferred into the proacrosomal vacuole and assembled into the acrosomal blastema. At the late spermatid, almost all of the basic protein in the nucleus has been removed into the acrosome. During the stage from late spermatid to mature sperm, some de novo basic proteins synthesized in the cytoplasm belt transfer into the nucleus without a membrane and almost all deposit in the periphery to form a supercoating. The remnant histone H4 accompanied by chromatin fibers is acetylated in the center of the nucleus, leading to relaxed DNA and activated genes making the nucleus non-condensed.     

细角螺卵子发生的细胞学和超微结构的研究

利用组织切片和透射电镜观察细角螺卵细胞发育的显微和超微结构,结果表明:细角螺卵原细胞期细胞核体积较大,呈椭圆形,核膜明显且有不规则的凹陷,细胞质内出现大量的线粒体和高尔基体.根据卵黄颗粒物的多少和大小可将卵母细胞分为前、中、后三个时期:前期卵母细胞细胞核内染色质浓缩,核仁可见,并出现核周间隙;中期卵母细胞内细胞核移向细胞的一端,核内染色质仍呈高电子密度状态,核仁不明显或消失;后期卵母细胞内的细胞核受挤压形状变得不规则,细胞质内可见少量的线粒体,大量的卵黄颗粒聚集在细胞质中并融合成很大的卵黄球.成熟期卵母细胞卵黄物质多且有较大的脂滴.     

Characterization of histone H2A and H2B variants and their post-translational modifications by mass spectrometry

The nucleosome, the fundamental structural unit of chromatin, contains an octamer of core histones H3, H4, H2A, and H2B. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function, analysis of histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2A and H2B variants derived from Jurkat cells. A combination of mass spectrometric techniques, HPLC separations, and enzymatic digestions using endoproteinase Glu-C, endoproteinase Arg-C, and trypsin were used to identify histone H2A and H2B subtypes and their modifications. We identified nine histone H2A and 11 histone H2B subtypes, among them proteins that only had been postulated at the gene level. The two main H2A variants, H2AO and H2AC, as well as H2AL were either acetylated at Lys-5 or phosphorylated at Ser-1. For the replacement histone H2AZ, acetylation at Lys-4 and Lys-7 was found. The main histone H2B variant, H2BA, was acetylated at Lys-12, -15, and -20. The analysis of core histone subtypes with their modifications provides a first step toward an understanding of the functional significance of the diversity of histone structures.     

Histone H4 acetylation and DNA methylation dynamics during pollen development

Summary The first pollen mitosis results in generative and vegetative cells which are characterised by a striking difference in their chromatin structure. In this study, histone H4 acetylation and DNA methylation have been analysed during pollen development inLilium longiflorum. Indirect immunofluorescence procedures followed by epifluorescence and laser scanning microscopy enabled a relative quantification of H4 acetylation and DNA methylation in microspores, immature binucleate pollen, mature pollen, and pollen tubes. The results show that histone H4 of the vegetative nucleus, in spite of its decondensed chromatin structure, is strongly hypoacetylated at lysine positions 5 and 8 in comparison with both the original microspore nucleus and the generative-cell nucleus. These H4 terminal lysines in the vegetative nucleus are, however, progressively acetylated during the following pollen tube growth. The DNA methylation analysis inversely correlates with the histone acetylation data. The vegetative nucleus in mature pollen grains is heavily methylated, but a dramatic nonreplicative demethylation occurs during the pollen tube development. Changes neither in H4 acetylation nor in DNA methylation have been found during development of the generative nucleus. The results obtained indicate that the vegetative nucleus enters the quiescent state (accompanied by DNA hypermethylation and H4 underacetylation) during the maturation of pollen grain which enables pollen grains a long-term survival without external source of nutrients until they reach the stigma.     

Isolation and characterization of acetylated histones H3 and H4 and their assembly into nucleosomes

Nucleosome and chromatin structure/function relationships of histone acetylations are not understood. To address these questions we have developed chromatographic procedures that separate subtypes of H3 and the acetylated states of histone H3 and H4 in exceptionally pure forms. The sites of acetylation of the intermediately acetylated states of H3 have been determined and show a specific pattern of acetylation. An unexpected finding was the identification of a fifth site of acetylation in H3 at lysine 27. Nucleosome particles with fully acetylated H3 and H4 have been assembled on the Lytechinus variegatus 5 S rRNA DNA phasing sequence and characterized. These defined acetylated H3 and H4 particles migrate more slowly in polyacrylamide nucleoprotein particle gels than the control particles indicating a subtle effect of acetylation in nucleosome structure. However, DNA footprinting of these particles using DNase I show only small changes when compared to control particles over the core particle DNA length. It is shown further that H3 cysteines in the particle containing fully acetylated H3 and H4 were not accessible to iodoacetamide indicating that protein factors additional to H3 and H4 acetylation are required to make H3 cysteines accessible to the label. These findings are consistent with the proposal that histones H3, H4 acetylations exert their major effects outside of the core particle 146-base pair DNA, either on the DNA segment entering and leaving the nucleosome or possibly on the internucleosome interactions that involve the amino-terminal domains of the core histones in organization and stability of higher order chromatin structures.     

Histone modifications related to chromosome silencing and elimination during male meiosis in Bengalese finch

We report here that a germline-restricted chromosome (GRC) is regularly present in males and females of the Bengalese finch (Lonchura domestica). While the GRC is euchromatic in oocytes, in spermatocytes this chromosome is cytologically seen as entirely heterochromatic and presumably inactive. The GRC is observed in the cytoplasm of secondary spermatocytes, indicating that its elimination from the nucleus occurs during the first meiotic division. By immunofluorescence on microspreads, we investigated the presence of histone H3 modifications throughout male meiosis, as well as in postmeiotic stages. We found that the GRC is highly enriched in di- and trimethylated histone H3 at lysine 9 during prophase I, in agreement with the presumed inactive state of this chromosome. At metaphase I, dimethylated histone H3 is no longer detectable on the GRC and its chromatin is more faintly stained with DAPI. The condensed GRC is underphosphorylated at serine 10 compared to the regular chromosomes during metaphase I, being phosphorylated later at this site after the first meiotic division. From these results, we proposed that trimethylation of histone H3 at lysine 9 on the GRC chromatin increases during metaphase I. This hypermethylated state at lysine 9 may preclude the phosphorylation of the adjacent serine 10 residue, providing an example of cross-talk of histone H3 modifications as described in experimental systems. The differential underphosphorylation of the GRC chromatin before elimination is interpreted as a cytologically detectable byproduct of deficient activity of Aurora B kinase, which is responsible for the phosphorylation of H3 at serine 10 during mitosis and meiosis.     

Phosphorylation protects sperm-specific histones H1 and H2B from proteolysis after fertilization

At intermediate stages of male pronucleus formation, sperm-derived chromatin is composed of hybrid nucleoprotein particles formed by sperm H1 (SpH1), dimers of sperm H2A-H2B (SpH2A-SpH2B), and a subset of maternal cleavage stage (CS) histone variants. At this stage in vivo, the CS histone variants are poly(ADP-ribosylated), while SpH2B and SpH1 are phosphorylated. We have postulated previously that the final steps of sperm chromatin remodeling involve a cysteine-protease (SpH-protease) that degrades sperm histones in a specific manner, leaving the maternal CS histone variants unaffected. More recently we have reported that the protection of CS histones from degradation is determined by the poly(ADP-ribose) moiety of these proteins. Because of the selectivity displayed by the SpH-protease, the coexistence of a subset of SpH together with CS histone variants at intermediate stages of male pronucleus remodeling remains intriguing. Consequently, we have investigated the phosphorylation state of SpH1 and SpH2B in relation to the possible protection of these proteins from proteolytic degradation. Histones H1 and H2B were purified from sperm, phosphorylated in vitro using the recombinant alpha-subunit of casein kinase 2, and then used as substrates in the standard assay of the SpH-protease. The phosphorylated forms of SpH1 and SpH2B were found to remain unaltered, while the nonphosphorylated forms were degraded. On the basis of this result, we postulate a novel role for the phosphorylation of SpH1 and SpH2B that occurs in vivo after fertilization, namely to protect these histones against degradation at intermediate stages of male chromatin remodeling.     

Different types of plant chromatin associated with modified histones H3 and H4 and methylated DNA

Eukaryotic chromosomes are organized into two large and distinct domains, euchromatin and heterochromatin, which are cytologically characterized by different degrees of chromatin compaction during interphase/prophase and by post-synthesis modifications of histones and DNA methylation. Typically, heterochromatin remains condensed during the entire cell cycle whereas euchromatin is decondensed at interphase. However, a fraction of the euchromatin can also remain condensed during interphase and appears as early condensing prophase chromatin. 5S and 45S rDNA sites and telomere DNA were used to characterize these regions in metaphase and interphase nuclei. We investigated the chromosomal distribution of modified histones and methylated DNA in the early and late condensing prophase chromatin of two species with clear differentiation between these domains. Both species, Costus spiralis and Eleutherine bulbosa, additionally have a small amount of classical heterochromatin detected by CMA/DAPI staining. The distribution of H4 acetylated at lysine 5 (H4K5ac), H3 phosphorylated at serine 10 (H3S10ph), H3 dimethylated at lysine 4 or 9 (H3K4me2, H3K9me2), and 5-methylcytosine was compared in metaphase, prophase, and interphase cells by immunostaining with specific antibodies. In both species, the late condensing prophase chromatin was highly enriched in H4K5ac and H3K4me2 whereas the early condensing chromatin was very poor in these marks. H3K9me2 was apparently uniformly distributed along the chromosomes whereas the early condensing chromatin was slightly enriched in 5-methylcytosine. Signals of H3S10ph were restricted to the pericentromeric region of all chromosomes. Notably, none of these marks distinguished classical heterochromatin from the early condensing euchromatin. It is suggested that the early condensing chromatin is an intermediate type between classical heterochromatin and euchromatin.